Atherosclerosis is a chronic disease that causes a thickening of the innermost layer (the intima) of large and medium-sized arteries. It decreases blood flow and may cause ischemia and tissue destruction in organs supplied by the affected vessel. Atherosclerosis is the major cause of cardiovascular disease including myocardial infarction, stroke and peripheral artery disease. It is the major cause of death in the Western world and is predicted to become the leading cause of death in the entire world within two decades.
Thus research during the last 20 years has shown that atherosclerosis is an inflammatory disease, which develops at sites of cholesterol accumulation in the artery wall. Specifically, activated T-cells macrophages and antibodies are found in atherosclerotic lesions of humans and experimental animals. A significant proportion of these T cells recognize oxidatively modified LDL (oxLDL). Such cells appear in increased amounts in the circulation of patients undergoing unstable angina and myocardial infarction. Similarly, antibodies to oxLDL can be detected in patient's blood and extracted from plaques. The importance of adaptive immunity for atherosclerosis is further underlined by the finding that severe combined immunodeficiency (SCID) leads to reduced atherosclerosis in hypercholesterolemic mice, while transfer of T cells into such mice increases the extent of the disease.
The LDL particle consists of several different molecules including triglycerides, cholesterol esters, phospholipids, and a large protein, apolipoprotein B100 (apoB). Oxidative modification results in lipid peroxidation, generation of reactive aldehydes, fragmentation of the protein, and binding of aldehydes to amino acid residues of the protein fragments. Antibody responses have been detected to several of these components, including phosphorylcholine and malondialdehyde-modified apoB protein and fragments thereof. Cellular immune responses are likely to be directed against modified protein components since the predominating components of cellular immunity found in lesions are CD4+T cells expressing TCRαβT cell receptors. These cells are known to recognize 15-25 amino acid peptides bound to Major Histocompatability Complex (MHC) class II proteins (e.g., Human Leucocyte Antigen (HLA-DR)) and it is therefore likely that such peptides serve as antigens for the cellular immune response associated with atherosclerosis. It is furthermore likely that we are tolerant to peptides of native apoB and that oxidation breaks tolerance, possibly through formation of aldehyde adducts.
Immunization with oxidized LDL has been shown to reduce atherosclerosis in experimental animals. This observation also suggests the possibility of developing an immune therapy or “vaccine” for treatment of atherosclerosis-based cardiovascular disease in man. One approach to do this would be to immunize an individual with his own LDL after it has been oxidized by exposure to for example copper. However, this approach is complicated by the fact that it is not known which structure in oxidized LDL that is responsible for inducing the protective immunity and if oxidized LDL also may contain epitopes that may give rise to adverse immune reactions.
The identification of epitopes in oxidized LDL is important for several aspects:
First, such epitopes are likely to be responsible for activating the anti-atherogenic immune response observed in animals immunized with oxidized LDL. Peptides containing these epitopes may therefore represent a possibility for development of an immune therapy or “atherosclerosis vaccine” in man. Further, they can be used for therapeutic treatment of atherosclerosis developed in man.
Secondly, peptides containing the identified epitopes can be used to develop ELISAs able to detect antibodies against specific structure in oxidized LDL. Such ELISAs would be more precise and reliable than those presently available using oxidized LDL particles as antigen. It would also allow the analyses of immune responses against different epitopes in oxidized LDL associated with cardiovascular disease.
U.S. Pat. No. 5,972,890 relates to a use of peptides for diagnosing atherosclerosis. The technique presented in said U.S. patent is as a principle a form of radiophysical diagnosis. A peptide sequence is radioactively labelled and is injected into the bloodstream. If this peptide sequence should be identical with sequences present in apoB it will bind to the tissue where there are receptors present for apoB. In vessels this is above all atherosclerotic plaque. The concentration of radioactivity in the wall of the vessel can then be determined e.g., by means of a gamma camera. The technique is thus a radiophysical diagnostic method based on the notion that radioactively labelled peptide sequences will bind to their normal tissue receptors present in atherosclerotic plaque and are detected using an external radioactivity analysis. It is a direct analysis method to identify atherosclerotic plaque. It requires that the patient be given radioactive compounds.
The technique of the present invention is based on quite different principles and methods. In accordance with claim 1 the invention relates to a fragment of apoB for immunisation against cardiovascular disease as well as a method for diagnosing immunological reactions against such a peptide sequence of apoB. Such immune reactions have in turn been found to be increased in individuals who have a developed atherosclerosis. The present technique is based on attaching the peptide sequence in the bottom of polymer wells. When a blood sample is added the peptide will bind antibodies, which are specific to this sequence. The amount of antibodies bound is then determined using an immunological method. In contrast to the technique of said U.S. patent this is thus not a direct determination method to identify and localise atherosclerotic plaque but determines an immunological response, which may show a high degree of co-variation with the extension of the atherosclerosis.
The basic principle of the present invention is thus quite different from that of said patent. The latter depends on binding of a peptide sequence to the normal receptors of the lipoproteins present in atherosclerotic tissue, while the former is based on the discovery of immune reactions against peptide sequence and determination of antibodies to the peptide sequence.
PCT/SE02/00679 discloses a number of peptides for peptide-based immunization therapy of atherosclerosis and development of a peptide-based assay for determination of immune responses against oxidized low density lipoprotein, said peptides being derived from apoB.
Published studies (Palinski et al., 1995, George et al., 1998 and Zhon et al., 2001) have shown that immunisation against oxidised LDL reduces the development of atherosclerosis. This would suggest that immuno reactions against oxidised LDL in general have a protecting effect. Previous results have (PCT/SE02/00679, above), however, surprisingly shown that this is not always the case. Thus in said reference it is shown that using a mixture of peptides #10, 45, 154, 199, and 240 will give rise to an increase of the development of atherosclerosis. Immunisation using other peptide sequences of said reference, e.g., peptide sequences #1, and 30 to 34 lacks total effect on the development of atherosclerosis. The results are surprising because they provide a basis for the fact that immune reactions against oxidised LDL, can protect against the development, contribute to the development of atherosclerosis, and be without any effect at all, depending on which structures in oxidised LDL they are directed to. These findings make it possible to develop immunisation methods, which isolate the activation of protecting immune reactions. Further, they show that immunisation using intact oxidised LDL could have a detrimental effect if the particles used contain a large amount of structures that give rise to atherogenic immuno reactions.
WO 99/08109 relates to the use of a panel of monoclonal mouse antibodies, which bind to particles of oxidised LDL in order to determine the presence of oxidised LDL in serum and plasma. This is thus totally different from the present invention wherein a method for determining antibodies against oxidised LDL is disclosed.
U.S. Pat. No. 4,970,144 relates to a method for preparing antibodies by means of immunisation using peptide sequences, which antibodies can be used for the determination of apolipoproteins using ELISA. This is thus something further quite different from the present invention.
U.S. Pat. No. 5,861,276 describes a recombinant antibody to the normal form of apolipoprotein B. This antibody is used for determining the presence of normal apolipoprotein B in plasma and serum, and for treating atherosclerosis by lowering the amount of particles of normal LDL in the circulation.
Thus in the present invention the use of antibodies are described for treating atherosclerosis. However, contrary to the U.S. Pat. No. 5,861,276, these antibodies are directed to structures present in particles of oxidised LDL and not to the normal particle of LDL. The advantage is that it is the oxidised LDL, which is supposed to give rise to the development of atherosclerosis. The use of antibodies directed to structures being specific to oxidised LDL is not described in said US patent.